I propose to define and characterize structural domains of rhodopsin in rod outer segment disc membranes and determine whether these parts of the molecule participate in particular biochemical functions. This will provide information about the location, extent, and structure of functional domains in rhodopsin. By integrating these two ways of studying the molecule I hope to more directly approach the question of what rhodopsin's specific roles are in visual excitation and control of photoreceptor sensitivity. Right sided and inverted disc membranes will be prepared and subjected to modification by different combinations of enzymatic proteolysis and cysteine specific cleavage. Cleaved fragments that are bound to the membrane through hydrophobic interactions will be dissociated. The modified disc membranes produced in these experiments will represent varying degrees of degradation and perturbation of rhodopsin structure and will be used to 1) identify regions of rhodopsin exposed at intradiscal and extradiscal surfaces 2) isolate transmembrane peptides and 3) determine the amount of secondary structure in rhodopsin's intrinsic membrane domain. Rhodopsin will also be modified by sulfhydryl reagents and transglutaminase labelling and hydrogen exchange kinetics will be used to evaluate the effects of modifications on the secondary structure of rhodopsin, fast photointermediates, and the apoprotein opsin. Modified disc membranes, both before and after illumination, will be tested for their ability to activate cyclic GMP phosphodiesterase or to stimulate phosphorylation of rhodopsin. Data from hydrogen exchange studies and the functional assays taken together will enable me to determine which parts of the rhodopsin are involved on the activation of phosphodiesterase. Because hydrogen exchange measurements detect discrete structure changes at different temporal stages in the bleaching cycle, it may also be possible to determine which intermediates of rhodopsin play a role in activation.